Mycalamide compounds, compositions thereof and methods of preparation and use

ABSTRACT

Mycalamide compounds are derived from marine sponges of the genus Mycale or prepared by synthetic methods. These compounds and pharmaceutical compositions containing them as active ingredients are useful as antitumor and antiviral agents.

REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of copending application U.S.Ser. No. 043,700, filed Apr. 29, 1987, now abandoned. The disclosure ofthat application is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to new organic compounds, novel biologicallyactive compositions containing one or more of such compounds and methodsfor the production of such compounds and compositions. Moreparticularly, the invention concerns novel compounds that possessantitumor, antiviral and other biologically active properties and theirmethods of production and use.

BACKGROUND OF THE INVENTION

Various tumor related diseases inflict man and other mammals and, asthese conditions are often fatal, the prevention, control of growth andregression of tumors in mammals has been receiving widespread attentionby the medical profession and pharmaceutical industry. The term tumorrefers to abnormal masses of new tissue growth which is discordant withthe economy of the tissue of origin or the host's body as a whole.

Tumors inflict mammals and man with a variety of disorders andconditions including various forms of cancer and resultant cancerouscachexia, which term refers to the symptomatic discomfort thataccompanies the infliction of a mammal with a tumor. Such symptomsinclude weakened condition of the inflicted mammal as evidenced byweight loss, etc. The seriousness of cancer is well known since canceris a major cause of death in man.

Considerable research and resources have been devoted to oncology andantitumor measures including chemotherapy. While various antitumoragents and methods have been developed which aid in inhibiting tumors,additional methods and chemical agents are needed.

Viral diseases also inflict man, plants, insects and animals. Theprevention and control of viral diseases has important health andeconomic implications.

Viral diseases contribute to inflictions in humans including commoncolds, herpes, acquired immune deficiency syndrome and cancer so theimportance of their control is obvious. Also important is the control ofviral diseases in animals for economic and other reasons, e.g., theability of such animals to become virus reservovirs or carriers whichfacilitate the spreading of viral diseases to humans. Viral plantdiseases have been known to have a disruptive effect on the cultivationof fruit trees, tobacco and various vegetables. Insect viral diseasesare also of interest because of the insects' ability to transfer viraldiseases to humans.

The prevention and control of viral diseases is thus of prime importanceto man and considerable research has been devoted to antiviral measures.Certain methods and chemical compositions have been developed which aidin inhibiting, controlling or destroying viruses, but additional methodsand antiviral compositions are needed.

A potential source for biologically active compounds of great diversityis marine plants and animals. In fact, marine sponges have proved to bea productive source for such compounds. Some such molecules derived fromsponges are described in Scheuer, P. J., Marine Natural Products,Chemical and Biological Perspectives; Academic Press; New York,1978-1983; Vol. I-V; Kato et al., Tetrahedron Letters, Vol. 26, Pg.3483-6 (1985); and Capon and Macleod, Tetrahedron, Vol. 41, Pg.3391-3404 (1985). The entire disclosures of these references are herebyincorporated herein by reference.

Another naturally derived composition of interest is pederin. Pederin isisolated from insects of the Paederus genus. Pederin shows antimitoticactivity as described in British Pat. Nos. 1,078,049 (1967) and 932,875(1963).

It has now been found that certain compounds derived from extracts ofmarine sponge of the genus Mycale, family Mycalidae, and orderPoecilosclerida, possess useful biological activity.

SUMMARY OF THE INVENTION

The invention provides new compounds having unique biologicalactivities, that have been given the class name mycalamides,compositions containing such compounds and methods for the preparationand for the use of the compounds and the compositions.

The full scope of applicability of the present invention will becomeapparent from the detailed description given hereinafter; it should beunderstood, however, that the detailed description, while indicatingpreferred embodiments of the invention, is given by way of illustrationonly, since various changes and modifications within the spirit andscope of the invention will become apparent to those skilled in the artfrom this detailed description

The objects of the invention are accomplished, in part, by the discoveryof novel biologically active compounds of the general formula (I):##STR1## R¹ and R² are the same or different and are hydrogen or loweralkyl, and

R³⁻⁶ are the same or different and are hydrogen, lower alkyl, carboxylicacyl or lower tri-(lower alkyl) silyl.

In preferred embodiments of the invention, the new compounds are insubstantially pure form.

In preferred embodiments of the invention R¹ and R² are methyl groups,R⁴ is hydrogen and Z is: ##STR2##

Also provided by discoveries of the invention are pharmaceuticalcompositions containing between about 0.01 to 50%/w of one of the newcompounds of the invention or a mixture of two or more of such compoundsand one or more non-toxic, compatible ingredient, e.g., carrier, diluentand/or adjuvant.

A preferred class of the new mycalamide compounds of the invention arethose having the formula: ##STR3## wherein R¹ and R² are the same ordifferent and are hydrogen or lower alkyl, particularly C1-C5 alkyl,

R³⁻⁶ are the same or different and are hydrogen, lower alkyl, acyl orlower alkyl silyl, and

X is ═CH₂, ##STR4##

The invention provides a variety of processes for the production ofcompounds of the invention. A preferred method of producing the newcompounds comprises the steps of collecting marine sponge of the genusMycale, family Mycalidae, and order Poecilosclerida, contacting suchsponge with a selected organic solvent system to obtain an extract,fractionating the extract and isolating mycalamide compounds of formulaI from the fractionated extract.

In further preferred methods of the invention, some of the newmycalamide compounds are made by hydrogenation in the presence of ahydrogenation catalyst. Also, ion-exchange, hydrolysis, alkylation,acetylation and other known synthesis type reactions may be usedpursuant to known procedures to add or modify various groups inpreferred compounds to produce other compounds according to the formulaI.

As a result of the discoveries by the invention of the new compounds,skilled chemists will be able to use known procedures to synthesizethese compounds from available stock substances.

The objects are further accomplished according to the invention by thediscovery that viruses and tumors can be inhibited by contact with aneffective amount of the new compounds.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A more complete understanding of the invention can be obtained byreference to preferred embodiments of the invention which areillustrated by the following specific examples of compounds,compositions and methods of the invention. It will be apparent to thoseskilled in the art that the examples involve use of materials andreagents that are commercially available from known sources, e.g.,chemical supply houses, so no details are given respecting them.

The following examples are not intended to be limiting of the scope ofthe invention, but to provide further understanding of the inventionincludingmethods for production of the mycalamide compounds,pharmaceutical compositions containing them and methods of using thecompounds and compositions.

EXAMPLE 1 Preparation of Mycalamide A

The compound of the formula: ##STR5##

A New Zealand maring sponge of the genus Mycale (family Mycalidae, orderPoecilosclerida), was collected from the channel at Aquarium Point,Otago Harbour, New Zealand. 200 gms. of frozen sponge were extracted byblendingwith 3:1 methanol:toluene and filtering off the solid. Onremoving the solvents, the combined extracts from three such stepsyielded a brown gum (11.04 gms) with antiviral properties. Reverse phaseflash chromatography (Blunt et al., J. Nat. Prod. 50, Pg290 (1987) gavebioactive fractions on eluting with 1:1, 3:1 and 9:1 mixtures of H₂O:methanol. These were combined to give a brown oil (307 mg). Asubsample of this material (140 mg) was applied to a column of FractogelPGM 2000 (120 gms Fractogel, column 43 cm×2 cm). Eluting with 1:4, H₂O:methanol (0.5 ml/min) gave bioactive fractions at around 1.5 voidvolumes, which were combined (brown oil, 50 mg). Silica gel columnchromatography (DAVISIL, 35-60u, 5 gms) starting with CH₂ Cl₂, thenincreasing amount of methanol, gave 1.7 mg of mycalamide A in the mostbioactive fraction, eluted with 1:9 methanol:CH₂ Cl₂.

Spectral data: [α] 365 nm+110° (c 0.2 gm/100 ml, CHCL₃)

MS (EI): M+, measured 50.27220 daltons, calculated for C₂₄ H₄₁ NO₁₀503.27305 (-1.7 ppm). M+-methanol, measured 471.24824 daltons,calculated for C₂₃ H₃₇ NO₉ 471.24683 (+3.0 ppm).

IR, film, (CM-1): 3700-3700, 2690, 1740, 1700, 1540, 1470, 1390, 1100,1080, 1040. ##STR6##

EXAMPLE 2 Preparation of Mycalamide B

The compound of the formula: ##STR7##

Mycalamide B, an oil having a molecular formula C₂₅ H₄₃ NO₁₀and amolecular weight of 517, was extracted, using the same general procedureas described in Example 1, from the same sponge, i.e., Mycale sp. (typespecimen PML1-9, Chemistry Department, University of Canterbury), familyMycalidae, order Poecilosclerida (Perry, et al. JACS, in press).Experience indicates that mycalamide A & B co-occur in all samples ofthe active Mycale sp. of sponge.

Spectral data: (α)_(D) +39° (c 0.2, CHCl₃).

HREIMS, M+--CH₃ O 486.26993 (-0.8 ppm), M+--CH₃ OH 485.26422 (+3.5 ppm).

DCIMS (NH₃) 535 (7%, M+NH₄ +), 505(28%), 504(38%), 503 (100% M+NH₄+--CH₃ OH), 488(23%), 487(36%), 486(89%).

DCIMS (ND₃) 543(5%), 542(14%), 541(10%), 513(17%), 493(8%), 492(9%),491(15%), 490(36%), 489(37%), 488(16%).

DCIMS (CH₄) 488(16%), 487(32%), 486(100%,MH+--CH₃ OH), 456(16%).

IR(film) 3700-3100, 2950, 1700, 1540, 1470, 1390, 1100, 1080, 1040,750cm⁻¹.

IR(CHCl₃) 3600-3300, 2900, 1690, 1390, 1100, 1040cm⁻¹.

¹ H NMR (CDCl₃) 7.54(NH9,d,10.0), 5.79(H10,t,9.7),5.12(10--O--CH₂,d,7.0), 4.85(10--O--CH₂,d,6.9), 4.85(4=CH₂,t,2.0),4.72(4=CH₂,t,1.9), 4.29(H7,s), 4.21(H12,dd,6.7,10.4),4.02(H2,dq,2.8,6.6), 3.79 (H11,dd,6.7,9.7), 3.65(H18,dd,3.3,11.9),3.55(13--O--CH₃,s), 3.47(H18,dd,5.7,11.9), 3.44(H13,d,10.5), 3.41(H15,dd,3.2,9.1), 3.29(6--O--CH₃,s), 3.24(17--O--CH₃,s), 3.2(H17,m),2.36(HS(eq),d,13.9), 2.22(H5(ax),td,2.0,13.9), 2.24(H3,dq,2,4,6.9),1.5(H₂ 16,m). 1.20(2--CH₃,d,6.6), 1.01(3--CH₃,d,7.1), 0.97(14--CH₃(eq),s), 0.85(14--CH₃ (ax),s) ppm(couplings in HZ).

C NMR (CDCL₃) 171.88(C8), 145.10(C4), 111.02(4=CH₂), 99.95(C6),86.49(10--O--CH₂), 79.27(C13), 78.84(C17), 75.46(C15), 74.44(C12),73.90(C10), 71.73(C7), 70.94(C11), 69.64(C2), 63.48(C18),61.78(13--O--CH₃) 56.64(17--O--CH₃), 48.57(6--O--CH₃), 41.47(C14),41.27(C3), 33.64(C5), 29.63(C16), 23.13(14--CH₃ (eq)), 17.93(2--CH₃),13.32(14--CH₃ (ax)), 12.13(3--CH₃).

AV assay: 4+, 4+, +*, C7 @2 ng/disk; -; -; - @0.5 ng/disk P388 IC₅₀ :0.26 ng/ml.

EXAMPLE 3 Preparation of Mycalamide A 4 γ-epoxide (major product)

The compound of the formula: ##STR8##

Mycalamide A (2.9 mg) and m-chloroperbenzoic acid (2 mg) were stirred inchloroform for 6 hours. The solvent was removed and the combined organicextract subjected to silica gel chromatography (200 mg Davisil, 150 A,35-70 um), developed in steps from 50% hexane in ethyl acetate to 10 %ethanol in ethyl acetate. Three fractions (2.3 mg combined) elutingbetween 5% and 10% ethanol in ethyl acetate contained mycalamide Aepoxide, a 2:1 mix of two isomers. These were separated by prep tlc(silica gel 60 F₂₅₄, 0.2 mm) developed twice with 10% ethanol in ethylacetate. The two bands of silica were recovered and each eluted with1:1EtOH:EtOAc giving two extracts (1.0 mg, 0.6 mg) which were epimers ofpure mycalamide A epoxide, an oil having a molecular formula C₂₄ H₄₁NO₁₁ and a molecular weight of 519.

Spectral data:

DCIMS (NH₃) 537 (10%, M+NH₄ ⁺), 505(22% M+NH₄ ⁺ --CH₃ OH), 488(17%,MH⁺--CH₃ OH), 487(15%), 335(34%), 262(16%), 231(24%), 230(34%).

¹ H NMR (CDCl₃) 7.40(NH9,d,9.7), 5.88(H10,t,9.8), 5.14(10--O--CH₂,d,7.0), 4.88(10--O--CH₂,d,7.0), 4.34(H₂,dq,2.4,6.6), 4.31(H7,s),4.24(H12,dd,6.8,10.4), 3.86(H11,dd,6.8,10.0), 3.74(H17,m),3.61(H15,dd,4.1,5.2) 3.56(H18,m,hidden), 3.56(13--O--CH₃,s),3.38(H18,dd,5.8,11.3), 3.37(6--O--CH₃,s), 3.47(H13,d,10.4), 2.55(4--CH₂O,d,4.5), 2.52(4--CH₂ O,d,4.5), 2.22(HS(ax,d,14.8), 1.55(H₂ 16,m),1.43(HS(eq),dd,1.3,14.8), 1.21(2--CH₃, d,6.6), 1.12(H3,m),1.00(3--CH₃,d,7.1), 0.98(14--CH₃ (eq),s), 0.88)14--CH₃ (ax),s)ppm(couplings in Hz).

C NMR (CDCl₃) 99.63(C6), 86.88(10--O--CH₂), 79.20(C13), 79.00(C15),74.47(C12), 73.62(C10), 73.28(C7), 71.65(C17), 71.42(C11), 67.18(C2),66.57(C18), 61.86(13--O--CH₃), 58.39(C4), 50.68(4--CH₂ O),49.05(6--O--CH₃), 41.74(C14), 39.71(C3), 31.93(C16), 31.40(C5),23.05(14--CH₃ (eq)), 17.53(2--CH₃), 13.37(14--CH₃ (ax)), 9.75(3--CH₃).

AV assay: 3+, 4+, +*, @200ng/disk; ++; +; +* @50 ng/disk.

P388 IC₅₀ : 34 ng/ml.

EXAMPLE 4 Preparation of Mycalamide A 4β-epoxide (minor product)

The compound of the formula: ##STR9##

Mycalamide A (2.9 mg) and m-chloroperbenzoic acid (2 mg) were stirred inchloroform for 6 hours. The solvent was removed and the combined organicextract subjected to silica gel chromatography (200 mg Davisil, 150 A,35-70 um), developed in steps from 50% hexane in ethyl acetate to 10%ethanol in ethyl acetate. Three fractions (2.3 mg combined) elutingbetween 5% and 10% ethanol in ethyl acetate contained mycalamide Aepoxide, a 2:1 mix of two isomers. These were separated by prep tlc(silica gel 60 F₂₅₄ 0.2 mm) developed twice with 10% ethanol inethylacetate. The two bands of silica were recovered and each elutedwith 1:1 EtOH:EtOAc giving two extracts (1.0 mg, 0.6 mg) which wereepimers of puremycalamide A epoxide, an oil having a molecular formulaC₂₄ H₄₁ NO₁₁ and a molecular weight of 519.

Spectral data:

DCIMS (NH₃) 537 (24%,M+NH₄ ⁺), 505(23% M+NH₄ ⁺ --CH₃ OH), 488(15%,MH⁺--CH₃ OH), 292(22%), 291(34%), 278(23%), 264(31%).

¹ H NMR (CDCl₃) 7.33(NH9,d,9.9), 5.88(H10,t,9.8),5.14(10--O--CH₂,d,7.0), 4.88(10--O--CH₂,d,7.0), 4.32(H7,2),4.24(H12,dd,6.6,10.5), 4.15(H2,dq,2.5,6.6), 3.86(H11,dd,6.7,10.0),3.73(H17,m), 3.63(H14,dd,4.0,5.6) 3.56(13--O--CH₃,s),3.55(H18,m,hidden), 3.47(H13,d,10.3), 3.39(H18,dd,6.6,11.0),3.32(6--O--CH₃,s), 2.79(4--CH₂ O,d,4.9), 2.71(4--CH₂ O,dd,1.6,4.8),2.10(HS(ax),dd,1.7,13.4), 1.55(H₂ 16,m), 1.43(HS(eq),dd,1.1,13.6),1.25(H3,m) 1.23(2--CH₃,d,6.6), 1.02(3--CH₃,d,7.0), 0.98(14--CH₃ (eq),s),0.88(14--CH₃ (ax),s) ppm(couplings in Hz).

¹³ C NMR (CDCl₃) 100.49(C6), 86.83(10--O--CH₂), 79.11(C13), 78.77(C15),74.46(C12), 73.77(C10), 72.58(C7), 71.19(C17), 71.29(C11), 68.80(C2),66.46(C18), 61.87(13--O--CH₃), 58.62(C4), 56.92(4--CH₂ O),48.60(6--O--CH₃), 41.71(C14), 39.97(C3), 32.23(C16,C5), 23.09(14--CH₃(eq), 17.74(2--CH₃) 13.41(14--CH₃ (ax)), 7.95(3--CH₃).

AV assay: 4+, 3+, +*, @ 50 ng/disk; 2+; +; +* @ 20 ng/disk.

P388 IC₅₀ : 14.9 ng/ml CB.

EXAMPLE 5 Preparation of Mycalamide B 4 α-epoxide (major product)

The compound of the formula: ##STR10##

Mycalamide B (6.8 mg) and m-chloroperbenzoic acid (6.5 mg) were stirredin chloroform for 2.5 days. The the combined organic extract was thentransferred directly onto a prep tlc plate (silica gel 60 F₂₅₄, 0.2 mm)and developed twice with 5% ethanol in ethyl acetate. Visualisationinvanillin/sulfuric acid show two separate brown bands at medium Rf. Thesilica was recovered and eluted with 1:1 EtOH:EtOAc giving two extracts.The solvent was removed and the two products (4 mg, 1.6 mg) were epimersof pure mycalamide A epoxide, an oil having a molecular formula C₂₅ H₄₃NO₁₁ and a molecular weight of 533.

Spectral data:

DCIMS (NH₃) 551 (14%, M+NH₄ ⁺), 519(23% M+NH₄ ³⁰ --CH₃ OH),502(36%,MH+--CH₃ OH), 409(12%), 386(15%), 351(15%), 349(15%), 306(23%),294(22%), 276(20%), 264(80%).

¹ H NMR (CDCl₃) 7.44(NH9,d,9.2), 5.77(H10,t,9.6),5.13(10--O--CH₂,d,7.0), 4.85(10--O--CH₂,d,7.0), 4.35(H2,dq,2.4,6.7),4.30(H7,s), 4.22(H12,dd,6.7,10.2), 3.82(H11,dd,6.7,9.7),3.62(H18,dd,3.1,12.0), 3.55(13--O--CH₃,s), 3.46(H18,dd,6.4,11.5),3.44(H13,d,10.2), 3.38(H15,dd,2.7,9.2), 3.37(6--O--CH₃,s),3.30(17--O--CH.sub.,s), 3.26(H17,m) 2.53(4--CH₂ O,d,4.6), 2.51(4--CH₂O,d,4.6), 2.13(HS(ax),d,14.8),1.56(H₂ 16,m), 1.44(HS(eq),dd,1.0,14.8),1.22(2--CH₃,d,6.9), 1.12(H3,m,), 1.01(3--CH₃,d,7.2), 0.98(14--CH₃(eq),s), 0.87 (14--CH₃ (ax),s) ppm(couplings in Hz).

¹³ C NMR (CDCL₃) 171.30(C8), 99.69(C6), 88.60(10--O--CH₂), 79.36(C13),79.24(C17). 75.36(C15), 74.48(C12), 73.77(C10), 72.81(C7), 70.83(C11),67.18(C2), 63.94(C18), 61.81(13--O--CH₃), 58.32(C4), 56.73(17--O--CH₃),50.51(4--CH₂ O), 48.87(6--OCH₃), 41.54(C14), 39.74(C3), 31.38(C5),29.47(C16), 23.16(14--CH₃ (eq)), 17.56(2--CH₃), 13.38(14--CH₃ (ax)),9.82(3--CH₃).

EXAMPLE 6 Preparation of Mycalamide B 4δ-epoxide (minor product)

The compound of the formula: ##STR11##

Mycalamide B (6.8 mg) and a m-chloroperbenzoic acid (6.5 mg) werestirred in chloroform for 2.5 days. The the combined organic extract wasthen transferred directly onto a prep tlc plate (silica gel 60 F₂₅₄, 0.2mm) and developed twice with 5% ethanol in ethyl acetate. Visualisationinvanillin/sulfuric acid show two separate brown bands at medium Rf. Thesilica was recovered and eluted with 1:1 EtOH:EtOAc giving two extracts.The solvent was removed and the two products (4 mg, 1.6 mg) were epimersof pure mycalamide A epoxide, an oil having a molecular formula C₂₅ H₄₃NO₁₁ and a molecular weight of 533.

Spectral data:

DCIMS (NH₃) 551 (17%, M+NH₄ ⁺), 542(16%), 528(50%), 519(32% M+NH₄ ⁺--CH₃ OH), 503(23%), 502(75%,MH+--CH₃ OH), 408(16%), 406(16%), 400(27%),391(18%), 386(58%), 373(22%), 372(100%).

¹ H NMR (CDCl₃) 7.43(NH9,d,9.9), 5.80(H10,t,9.6), 5.13(10--)CH₂,d,7.0),4.86(10--O--CH₂,d,6.9), 4.31(H7,s), 4.23(H12,dd,6.8,10.4),4.18(H2,dq,2.6,6.7), 3.81(H11,dd,6.8,9.7), 3.64(H18,m),3.56(13--O--CH₃,s), 3.44(H18,m,hidden), 3.43(H13,d,10.2), 3.38(H15,M),3.32(6--O--CH₃,s), 3.32(17--O--CH₃,s), 3.24(H17,m) 2.78(4--CH₂ O,d,4.9),2.69(4--CH₂ O,dd,1.5,4.9), 2.03(H5(ax),dd,1.5,13.2), 1.56(H₂ 16,m),1.42(H5(eq),dd,0.8,13.3), 1.25(H3,m) 1.24(2--CH₃, d,6.7),1.04(3--CH₃,d,7.0), 0.99(14--CH₃ (eq),s), 0.88(14--CH₃ (ax),s)ppm(couplings in Hz).

¹³ C NMR (CDCl₃) 100.72(C6), 86.53(10--O--CH₂), 79.36(C13), 78.78(C17).75.60(C15), 74.48(C12), 73.98(C10), 71.62(C7), 68.84(C2), 63.36(C18),61.82(13--O--CH₃), 58.42(C4), 56.76(4--CH₂ O),56.59(17--O--CH₃),48.37(6--O--CH₃), 41.74(C14), 40.09(C3), 32.00(C5),29.75(C16), 23.22(14--CH₃ (eq)), 17.80(2--CH₃), 8.06(3--CH₃). NB. C8,C11 and 14--CH₃ (ax) not observed.

EXAMPLE 7 Mycalamide A Triacetate

The compound of the formula: ##STR12##

Mycalamide A (2 mg) was dissolved in pyridine (0.5 ml) and aceticanhydride(0.5 ml). After 7 hr. at 21° C., water (1 ml) was added and themixture extracted with CHCl₃ (3×1 ml). The solvent was removed and thecombined organic extracts were subjected to silica gel chromotography(200 mg Davisil, 150 A, 35-70 um), developed in steps from hexane toethyl acetate. A fraction (1.2 mg) eluted with 1:1 hexane:ethyl acetatewas pure mycalamide triacetate, an oil of molecular weight 629andmolecular formula C₃₀ H₄₇ NO₁₃.

Spectral data:

HREIMS M+--CH₃ O 598.2800(-10.6 ppm), M+--CH₃ OH 597.27536(-5.3 ppm).

DCIMS (NH₃) 647 (13%, M+NH₄), 617(29%), 616(37%), 615(100%,M+NH₄ +--CH₃OH), 542(35%), 318(42%), 317(44%), 299(30%), 286(58%), 285(41%),270(28%), 269(60%), 257(30%).

DCIMS (CH₄) 598(71%,MH+--CH₃ OH), 538(100%,MH+--CH₃ OH--CH₃ CO₂ H),299(53%), 269(60%), 240(41%), 208(55%).

IR (CHCl₃) 3380, 2950, 2870, 1745, 1380, 1100, 1030 cm⁻¹

¹ H NMR (CDCl₃) 7.32(NH9,d,9.4), 5.76(H10,t,9.0), 5.47(H7,s),5.06(10--O--CH₂,d,6.9), 4.98(H17,m), 4.87(4=CH₂,m),4.86(10--O--CH₂,d,7.0), 4.76(4=CH₂,m), 4.27(H18,dd,2.7,12.4),4.14(H18,dd,5.2,12.4), 4.10(H12,dd,6.0,9.4), 3.99(H2,d1,2.8,6.6),3.79(H11,dd,6.0,8.7), 3.52(13--O--CH₃,s), 3.36(H13,d,9.4),3.45(H15,dd,2.4,9.8), 3.18(6--O--CH₃,s), 2.4(H₂ 5,m),2.25(H3,dq,2.7,7.0), 2.20(7--O.CO.CH₃,s), 2.05 and 2.00(17--O.CO.CH₃,sand 18--O.CO.CH₃,2xs), 1.7-1.8(H₂ 16,m), 1.20(2--CH₃,d,6.6), 1.03(3--CH₃,d,7.1), 1.01(14--CH₃ (eq),s), 0.86(14--CH₃ (ax),s) ppm(couplingsin Hz).

¹³ C NMR (CDCL₃) 170.60(C8), 169.85 & 167.64 & 167.01(7--O--CO,17--O--CO & 18--O--C), 145.08(C4), 110.85(4=CH₂), 99.26(C6),86.53(10--O--CH₂), 79.73(C13), 75.53(C15), 74.06(C12), 73.72(C10),71.66(C7), 70.01(C17), 69.91(C11), 69.73(C2), 63.57(C18),61.55(13--O--CH₃), 48.60(6--O--CH₃), 41.24(C14,C3), 34.18(C5),30.08(C16), 23.69(14--CH₃ (eq)), 21.06 & 20.80 & 20.65(7--, 17--, &18--O--COCH₃), 17.87(2--CH₃), 14.4(14--CH₃ (ax)), 12.04(3--CH₃ )ppm.

AV assay: 3+, 3+, +*, @ 1000 ng/disk; -, -, - @ 250 ng/disk.

P388 IC₅₀ : 51.9 ng/ml.

EXAMPLE 8 Mycalamide A Tripropanoate

The compound of the formula: ##STR13##

Mycalamide A (2.8 mg) was dissolved in pyridine (0.2 ml) and propionicanhydride (0.1 ml) and stirred overnight at room temperature. Water (0.2ml) was added and the mixture extracted with CHCl₃ (3×0.4 ml). Thesolvent was removed to give 3.7 mg of pure mycalamide tripropanoate, anoil of molecular weight 671 and molecular formula C₃₃ H₅₃ NO₁₃.

Spectral data:

DCIMS (NH₃) 689(20%, M+NH₄), 657(24%,M+NH₄ +--CH₃ OH), 404(23%),346(52%), 331(44%), 314(28%), 244(85%).

¹ H NMR (CDCl₃) 7.34(NH9,d,9.5), 5.77(H10,t,9.0), 5.48(H7,s),5.06(10--O--CH₂,d,7.0), 4.97(H17,m), 4.85(4=CH₂,m),4.85(10--O--CH₂,d,6.8), 4.75(4=CH₂,t,1.7), 4.28(H18,dd,2.8,12.4),4.16(H18,dd,5.2,12.4), 4.12(H12,dd,6.0,9.4), 3.99(HZ,d1,2.8,6.6),3.79(H11,dd,6.1,8.7), 3.52(13--O--CH₃,s), 3.45(H15,dd,2.5,9.8),3.37(H13,d,9.4), 3.17(6--O--CH₃,s), 2.49. 2.37 & 2.29 (7--O.CO.CH₂,17--O.CO.CH₂ &18--O.CO.CH₂,m), 2.4(H₂ 5,hidden), 2.3(H3,hidden),1.77(H16,ddd,2.4,9.3,14.3), 1.6-1.7(16,m), 1.19(2--CH₃,d,6.6),1.0-1.21(7-- ,17--&18--O.CO.CH₂ CH₃,m), 1.02(3--CH₃,d,7.1), 0.99(14--CH₃(eq),s), 0.85(14--CH₃ (ax),s) ppm(couplings in Hz).

¹³ C NMR (CDCL₃) 173.95,173.21&173.12(7--,17--&18--O--CO), 145.15(C4),110.77(4=CH₂), 99.32(C6), 86.52(10--O--CH₂), 79.70(C13), 75.45(C15),74.03(C12), 73.78(C10), 71.50(C7), 69.96(C17,C11),69.61(C2), 63.25(C18),61.56(13--O--CH₃), 48.60(6--O--CH₃), 41.25(C14,C3), 34.26(C5),30.10(C16), 27.55, 27.46 & 27.34(7--,17--&18--O--COCH₂), 23.65(14--CH₃(eq)), 17.88(2--CH₃), 12.03(3--CH₃), 9.11, 9.02 &8.90(7--,17--&18--OCOCH₂ CH₃)ppm. NB. C8, 14--CH₃ (ax) not observed.

AV assay: 4+, 4+, +*, @ 500 ng/disk; +, -, - @ 200 ng/disk;

P388 IC₅₀ : 85.6 ng/ml

EXAMPLE 9 Mycalamide B Diacetate

The compound of the formula: ##STR14##

Mycalamide B (3.5 mg) was dissolved in pyridine (0.1 ml) and aceticanhydride (0.1 ml). After 4 hr. at 21° C., water (0.2 ml) was addedandthe mixture extracted with CHCl₃ (3×0.2 ml). The solvent wasremoved andthe combined organic extracts were subjected to silica gelchromotography (200 mg Davisil, 150 A, 35-70 um), developed in stepsfrom hexane to ethyl acetate. A fraction (3 mg) eluted with 1:1hexane:ethyl acetate was pure mycalamide diacetate, an oil of molecularweight 601 and molecular formula C₂₉ H₄₇ NO₁₂.

Spectral data:

DCIMS (NH₃) 619(22%, M+NH₄), 589(16%), 588(32%), 587(100%,M+NH₄ +--CH₃OH), 570(25%,MH+--CH₃ OH), 318(19%), 290(25%), 258(62%), 257(26%),241(45%).

IR (CHCL₃) 3400, 2950, 2900, 1750, 1710, 1380, 1100, 1030, 910 cm⁻¹.

¹ H NMR (CDCl₃) 7.27(NH9,d,9.4), 5.75(H10,t,9.4), 5.45(H7,s),5.07(10--O--CH₂, d,7.0), 4.88(4=CH₂,m), 4.85(10--O--CH₂,d,6.9),4.75(4=CH₂,m), 4.28(H18,dd,2.5,12.3), 4.17(H12,dd,6.6,10.2),4.07(H18,dd,4.7,12.5), 4.02(H2,dq,2.8,6.7), 3.77(H11,dd,6.5,9.4),3.53(13--O--CH₃,s), 3.39(H13,d,9.8), 3.3(H17,m), 3.3(H15,hidden),3.25(17--O--CH₃,s), 3.17(6--O--CH₃,s), 2.4(H₂ 5,m), 2.28(H3,dq,2.7,7.3),2.20(7--O.CO.C₃,s), 2.08(18--O.C.CH₃,s), 1.6-1.7(H₂ 16,m),1.22(2--CH₃,d,6.5), 1.04(3--CH₃,d,7.2), 0.97(14--CH₃ (eq),s),0.86(14--CH₃ (ax),s) ppm(couplings in Hz).

⁻⁻ C NMR (CDCL₃) 170.85(C8), 169.68 & 166.67(7--&18--O--CO), 144.75(C4),111.26(4=CH₂), 99.15(C6), 86.53(10--O--CH₂), 79.43(C13), 77.93(C17),75.68(C15), 74.18(C12), 74.02(C10), 71.48(C7), 70.64(C11), 69.95(C2),63.48(C18), 61.70(13--O--CH₃), 56.88(17--O--CH₃), 48.49(6--O--CH₃),41.24(C14), 41.16(C3), 34.39(C5), 30.33(C16), 23.38(14--CH₃ (eq)), 20.97& 20.65(7--O--COCH₃, & 18--O--COCH₃), 17.92(2--CH₃), 13.8(14--CH₃(ax),broad), 12.19(3--CH₃)ppm.

AV assay: 4+, 4+, +*, @ 1000 ng/disk; -, -, - @ 250 ng/disk.

P388 IC₅₀ : 15.3 ng/ml.

EXAMPLE 10 Mycalamide A 7-mono-p-bromobenzoate.

The compound of the formula: ##STR15##

Mycalamide A (2.7 mg), p-bromobenzoyl chloride (5 mg) and triethylamine(2.9 mg) were stirred in CH₂ CL₂ at room temperature for 24 hours.Column chromatography on Davisil (0.2 g) gave a mixture of two products(2 mg ca.) and unreacted mycalamide A (0.8 mg.). Prep RPLC (20% H₂ O inMeOH) gave two fractions (1.3 mg, 0.5 mg) which were pure mycalamide Amono-p-bromobenzoates at C18--O and C7--O respectively, the latter beinga white solid having a molecular weight of 686 and a molecular formulaC₃₁ H₄₄ BrNO₁₁.

Spectral data:

¹ H NMR (CDCl₃) 7.96(7--OCOC₆ H₄ Br,d,8.8), 7.62(7--OCOC₆ H₄ Br,d,8.7),7.40(NH9,d,9.5), 5.83(H10, t,9.6), 5.70(H7,s,), 5.10(10--OCH₂,d,6.9),4.90(4=CH₂,m), 4.89(10--OCH₂,d,7.0), 4.82(4=CH₂,m),4.21(H12,dd,7.1,10.4), 4.05(H2,dq,2.7,6.4), 3.79(H11,dd,6.8,10.0),3.70(H17,m), 3.60(H15,m), 3.54(13--OCH₃,s), 3.47(H18,m),3.41(H13,d,10.2), 3.29(H18,dd,7.1,11.5), 3.21(6--OCH₃,s), 2.64(H5(ax),broad d,14.1), 2.46(H5(eq),d,14.6), 2.30(H3,dq,2.8,6.9), 1.5(H₂ 16,m),1.24(2--CH₃,d,6.5), 1.06(3--CH₃ ,d,7.2), 0.93(14--CH₃ (eq),s),0.85(14--CH₃ (ax),s) ppm(couplings in Hz).

AV assay: 4+, 4+, +*, @ 200 ng/disk, -, +, +* @ 50 ng/ml P388 IC₅₀ 23.3ng/ml.

EXAMPLE 11 Mycalamide A 18-mono-p-bromobenzoate.

The compound of the formula: ##STR16##prepared as stated in Example 10is a white solid having a molecular weightof 686 and a molecular formulaC₃₁ H₄₄ BrNO₁₁.

Spectral data:

¹ H NMR (CDCl₃) 7.89(18--OCOC₆ H₄ Br,d,8.6), 7.57(18--OCOC₆ H₄Br,d,8.7), 7.49(NH9,d,10.2), 5.88(H10, t,9.6),5.14(10--OCH₂,d,6.9),4.88(10--OCH₂,d,6.9), 4.83(4=CH₂,t,1.6), 4.68(4=CH₂,t,1.0),4.28(H7d,2.6), 4.23(H₂ 18,m), 4.23(H12,dd,6.5,10.5), 4.03(H17,m),3.98(H2,dq,2.7,6.6), 3.86(H11,dd,6.6,9.8), 3.76(C7--OH,d,2.6),3.71(H15,dd,2.3,9.9), 3.56(13--OCH₃,s), 3.47(H13,d,10.3),3.27(6--OCH₃,s), 3.20(C17--OH,m), 2.4(H₂ 5,m), 2.23(H3,dq,2.6,6.9),1.5-1.7(H₂ 16,m), 1.18(2--CH₃,d,6.6), 1.00(14--CH₃ (eq),s),0.98(3--CH₃,d,7.0), 0.90(14--CH₃ (ax),s) ppm(couplings in Hz).

AV assay: 3+, 4+, +*, @ 2 ng/disk; -, -, -, @ 0.5 ng/disk

P388 IC₅₀ 1.28 ng/ml.

EXAMPLE 12 Mycalamide A 7,18-di-p-bromobenzoate

The compound of the formula: ##STR17##

Mycalamide A (11 mg), p-bromobenzoyl chloride (23 mg) and triethylamine(10mg) were stirred in CH₂ CL₂ at room temperature for 24 hours. Columnchromatography on Davisil (0.2 g) gave a mixture of five products (12 mgca.). Prep RPLC (15% H₂ O in MeOH) gave five fractions (0.7 mg, 2.7 mg,1.2 mg, 2.5 mg, 2.7 mg) which were the two mycalamide Amono-p-bromobenzoates at C18--O and C7--O, two isomers of less saturatedmycalamide A dibenzoates (named neomycalamide A 7,18-dibenzoate) andmycalamide A 7,18-di-p-bromobenzoate, which is a white solid having amolecular weight of 869 and a molecular formula C₃₈ H₄₇ Br₂No₁₂.

Spectral data:

¹ H NMR (CDCl₃) 7.91 & 7.89(7--&18--OCOC₆ H₄ Br,d,8.5),7.56 &7,51(7--&18--OCOC₆ H₄ Br,d,8.6), 7.45(NH9,d,9.1), 5.85(H10, t,9.4),5.7(H7,s), 5.11(10--OCH₂,d,6.9), 4.90(10--OCH₂,d,6.9),4.88(4═CH₂,t,1.9), 4.72(4═CH₂,t,1.8), 4.21(H12,dd,6.3,10.9), 4.2(H₂18,m), 4.04(H2,dq,2.7,6.7), 4.03(H17,m), 3.82(H11,dd,6.7,9.7),3.76(H15,dd,2.7,9.7), 3.55(13--OCH₃,s), 3.44(H13,d,10.5),3.20(6--OCH₃,s), 2.69(H5(ax),broad d,14.4), 2.42(H5(eq),d.14.4),2.30(H3,dq,2.8,7.0), 1.6-1.7(H₂ 16,m), 1.23(2--CH₃,d,6.6),1.05(3--CH₃,d.7.0), 0.96(14--CH₃ (eq),s), 0.88(14--CH₃ (ax),s)ppm(couplings in Hz).

¹³ C NMR (CDCl₃) 167.22 & 165.72(7--&18--O--CO), 144.67(C4), 131.82,131.74, 131.50, 131.28, 131.14, 129.04, 128.74 & 128.17(7--&18--OCOC₆ H₄Br), 111.24(4=CH₂), 99.43(C6), 86.88(10--O--CH₂), 79.06(C13),79.03(C15), 74.54(C12), 74.31(C10), 72.10(C7), 71.58(C11), 70.18(C17),68.82(C2), 68.45(C18), 61.80(13--O--CH₃), 48.61(6--O--CH₃), 41.62(C14),41.23(C3), 34.24(C5), 32.81(C16), 23.20(14--CH₃ (eq)), 17.89(2--CH₃),13.66(14--CH₃ (ax)), 12.14(3--CH₃)ppm. NB. C8 not observed.

AV assay: 4+, 4+, +*, @ 2000 ng/disk; +, 2+, +* @ 5000 ng/disk

P388 IC₅₀ : 141.2 ng/ml.

EXAMPLE 13 Neomycalamide A 7,18-di-p-bromobenzoate

The compound of the formula: ##STR18##

From the preparation of mycalamide A di-p-bromobenzoate in Example 12,two further mycalamide analogs were separated and analysed by PMR. Theseresult from the loss of methanol across C6-C7 and migration of theexocyclic double bond at C4 to C4-C5 position under slightly acidicconditions. The first isomer was unstable and changed overnight in CDCL₃solution into the second, which differs only in the stereochemistry atthe new C6-C7 double bond and appears to be a stable white solid ofmolecular weight 837 and a molecular formula of C₃₇ H₄₃ Br₂ No₁₁.

Spectral data:

¹ H NMR (CDCl₃) 8.09(NH9,d,9.0),7.95 & 7.87(7--&18--OCOC₆ H₄ Br,d,8.6),7.56 & 7.50(7--&18--OCOC₆ H₄ Br,d,8.6), 5.99(H10),t,9.7),5.97(HS,q,1.5), 5.15(10--OCH₂,d,6.8), 4.88(10--OCH₂,d,7.0),4.33(H2,dq,2.8,6.4), 4.26(H₂ 18,m), 4.25(H12,m,hidden), 4.07(H17,m),3.89(H11,dd,6.7,10.2), 3.67(H15,broad d,9.6), 3.55(13--OCH₃,s),3.48(H13,d,10.6), 2.06(H3,dq,2.8,7.2), 1.83(4--CH₃, d,1.5), 1.6-1.7(H₂16,m), 1.41(2--CH₃,d,6.5), 0.99(3--CH₃,d.7.0), 0.95(14--CH₃ (eq),s),0.89(14--CH₃ (ax),s) ppm(couplings in Hz).

AV assay: 4+, 4+, +*, @ 2000 ng/disk; -, -, - @ 500 ng/disk

P388 IC₅₀ : 572.5 ng/ml.

EXAMPLE 14 Mycalamide A 17,18-bis-trimethylsilylether

The compound of the formula: ##STR19##

Mycalamide A (1 mg), was dissolved in pyridine (0.1 ml) and cooled inice. BSA (N,O-bistrimethylsilyl acetamide) (0.1 ml) was added and thereaction was allowed to proceed for four minutes. Water (0.5 ml) wasthen added andthe mixture extracted with CH₂ Cl₂ (30×0.2 ml). Thesolventwas removed and the combined organic extracts were subject tosilica gel chromatography (200 mg Davisil, 150 A, 35-70 um), developedin steps from hexane to ethyl acetate. A fraction (1.3 mg) eluted with2:1 hexane:ethyl acetate was pure mycalamide A17,18-bis-trimethylsilylether by PMR, being an oil having a molecularweight of 647 and a molecular formula C₃₀ H₅₇ NO₁₀ Si₂.

Spectral data:

HREIMS M+--CH₃ O 616.3292 (-7.3 ppm), M+--CH₃ OH 615.3263 (+0.7 ppm).

¹ H NMR (CD₂ Cl₂) 7.54(NH9,d,9.6), 5.72(H10),t,9.1),5.12(10--OCH₂,d,7.0), 4.87(4=CH₂,t,1.8), 4.83(10--OCH₂,d,7.0),4.71(4=CH₂,t,1.8). 4.22(H7,d,2.6), 4.14(H12,dd,6.3,9.5),3.99(H2,dq,2.8,6.6), 3.94(7--OH,d,2.6), 3.82(H11,dd,6.5,9.0),3.73(H17,m), 3.53(13--OCH₃,s), 3.52(H₂ 18,m), 3.42(H13,d,10.0),3.35(H15,dd,2.1,10.1), 3.28(6--OCH₃,s), 2.34(H5(eq),d,14.0),2.27(H3,dq,2.3,6.8), 2.24(H5(ax),td,2.2,14.0),1.71(H16,ddd,2.0,9.7,14.4), 1.45(H16,m) 1.21(2--CH₃,d,6.6),1.02(3--CH₃,d,7.3), 1.01(14--CH.sub. 3 (eq),s), 0.86(14--CH₃ (ax),s),0.14(18--OSi(CH₃)₃,s), 0.06(17--OSi(CH₃)₃,s),ppm(couplings in Hz).

AV assay: 4+, 4+, +*, @ 20 ng/disk; 2+, 3+, +* @ 5 ng/disk

P388 IC₅₀ : 1.2 ng/ml.

EXAMPLE 15 Mycalamide A tris-trimethylsilylether

The compound of the formula: ##STR20##

Mycalamide A (2.5 mg), was dissolved in pyridine (0.1 ml) and BSA(N,O-bistrimethylsilyl acetamide) (0.1 ml) was added and the reactionmixture was stirred overnight at room temperature. Water (0.3 ml) wasadded and the mixture extracted with CH₂ Cl₂ (3×0.2 ml). The solvent wasremoved and the combined organic extracts were subject to silica gelchromatography (200 mg Davisil, 150 A, 35-70 um), developed in stepsfrom hexane to ethyl acetate. A fraction (3 mg) eluted with 4:1hexane:ethyl acetate was pure mycalamide A tris-trimethylsilylether,beingan oil having a molecular weight of 719 and a molecular formula C₃₃H65NO₁₀ SI₃.

Spectral data:

HREIMS M+--CH₃ O 688.3742 (+1.4 ppm), M+--CH₃ OH 687.3651 (-0.4 ppm).

¹ H NMR (CDCl₃) 7.38(NH9,d,9.6), 5.76(H10,t,9.7), 5.11(10--OCH₂,d,6.9),4.81(10--OCH₂,d,6.9), 4.81(4-CH₂,t,2.0), 4.71(4=CH₂,t,2.1), 4.25(H7,s),4.17(H12,dd,6.4,10.2), 3.88(H2,dq,2.7,6.6), 3.77(H11,dd,6.4,9.5),3.72(H17,m), 3.54(13--OCH₃,s), 3.53(H18,dd,2.4,12.1),3.47(H18,dd,4.4,11.4), 3.42(H13,d,10.3), 3.34(H15,dd,1.7,9.9),3.28(6--OCH₃,s), 2.58(H5(eq),d,14.5), 2.35(HS(ax),td,1.9,14.7),2.19(H3,dq,2.6,6.8), 1.74(H16,ddd,2.0,10.3,13.8), 1.43(H16,m)1.18(2--CH₃,d,6.6), 1.00(3--CH₃,d.7.0), 0.98(14--CH₃ (eq),s),0.86(14--CH₃ (ax),s), 0.21(7--OSi(CH₃)₃,s), 0.10 &0.06(17--,18--OSi(CH₃)₃,s), ppm(couplings in Hz).

¹ H NMR (CD₂ Cl₂) 7.40(NH9,d,9.8), 5.73(H10,t,9.6),5.13(10--OCH₂,d,7.1), 4.81(10--OCH₂,d,7.0), 4.81(4=CH₂,t,2.2),4.68(4=CH₂,t,2.0), 4.23(H7,s), 4.15(H12,dd,6.5,9.8),3.86(H2,dq,2.6,6.6), 3.78(H11,dd,6.4,9.3), 3.73(H17,m),3.54(13--OCH₃,s), 3.53(H18,m), 3.48(H18,dd,4.4,11.8), 3.44(H13,d,10.0),3.33(H15,dd,1.9,10.1), 3.27(6--OCH₃,s), 2.54(HS(eq),d,14.2),2.35(HS(ax),td,2.1,14.4), 2.21(H3,dq,2.7,7.0),1.75(H16,ddd,2.0,10.0,14.0), 1.43(H16,m) 1.19(2--CH₃,d,6.6),1.01(3--CH₃,d.7.0), 1.01(14--CH₃ (eq),s), 0.86(14--CH.sub. 3 (ax),s),0.22(7--OSi(CH₃)₃,s), 0.12 & 0.07(17--, 18--OSi(CH₃)₃,s), ppm(couplingsin Hz).

¹³ C NMR (CD₂ Cl₂) 167.95(C8), 147.37(C4), 109.57(4=CH₂), 99.51(C₆),86.48(10--O--CH₂), 79.75(C13), 77.70(C15), 76.00(C7), 74.35(C17),73.37(C10), 70.67(C11, braod), 70.27(C13), 69.66(C2), 65.28(C18),61.57(13--O--CH₃) 50.05(6l --O--CH₃), 41.59(C3), 41.37(C14), 36.11(C5),33.36(C16), 23.40(14--CH₃ (eq)), 17.85(2--CH₃), 13.89(14--CH₃ (ax)),11.78(3--CH₃) and 0.31, 0.27 & -0.39(7--, 17--&18--OSi(CH₃)₃).

AV assay: 4+, 4+, +*, @ 20 ng/disk; -, -, - @ 5 ng/disk

P388 IC₅₀ : 1.3 ng/ml.

EXAMPLE 16 Mycalamide A 18-mono-t-butyl,dimethylsilylether

The compound of the formula: ##STR21##

Mycalamide A (1 mg), t-butyldimethylchlorosilane (8 mg),dimethylaminopyridine (1 mg) and trimethylamine (4 mg) were stirred inpyridine (0.2 ml) at room temperature for three hours. Water (0.5 ml)was added and the mixture extracted with CH₂ Cl₂. The organic extractwas washed with water (3×0.3 ml), the solvent was removed and thecombined product subjected to silica gel chromatography (200 mg Davisil,150 A, 35-70 um), developed in steps from hexane to ethyl acetate. Themajor fraction (0.8 mg) eluted with 2:1 hexane:ethyl acetate was puremycalamide A 18-mono-TBDMS ether, an oil having a molecular weight of617 and a molecular formula C₃₀ H₅₅ NO₁₀ Si.

Spectral data:

¹ H NMR (CD₂ Cl₂) 7.46(NH9,d,9.7), 5.83(H10,t,9.7),5.14(10--OCH₂,d,7.0), 4.86(10--OCH₂,d,-b 7.0), 4.85(4=CH₂ m),4.71(4=CH₂,m), 4.25(H7,d,2.6), 4.17(H12,dd,6.6,10.2),3.99(H2,dq,2.8,6.6), 3.84(H11,dd,6.4,9.2), 3.79(7--OH,d,2.6),3.62(H17,m),3.54(13--OCH₃,s), 3.53(H18,m), 3.5(H15,hidden), 3.47(H18,m),3.46(H13,d,hidden), 3.28(6--OCH₃,s), 2,80(17--OH,d,2.8), 2.30(H5,m),2.26(H3,dq,2.6,7.1), 1.73(H16,m), 1.5(H16,m) 1.20(2--CH₃,d,6.6),1.01(3--CH₃, d.7.1), 1.01(14--CH₃ (eq),s), 0.90(SiC(CH₃)₃,s),0.88(14--CH₃ (ax),s), 0.08(18--OSi(CH₃)₂,s) ppm(couplings in Hz).

¹³ C NMR (CD₂ Cl₂) 110.19(4=CH₂), 99.99(C6), 86.88(10--O--CH₂),79.57(C13), 79.07(C15), 74.40(C12), 74.08(C10), 72.61(C7), 71.37(C17),69.82(C2), 66.54(C18), 61.66(13--O--CH₃) 48.76(6--O--CH₃), 41.60(C3),33.83(C5), 32.36(C16), 25.84(18--OSiC(CH₃)₃), 23.27(14--CH₃ (eq)),17.81(2--CH₃), 13.90(14--CH₃ (ax)), 12.09(3--CH₃), -5.48(18--OSi(CH₃)₂).NB. C4, C8, C11, C14, 18--OSiC not observed.

AV assay: 4+, 4+, +*, @ 20 ng/disk; -, -, - @ 5 ng/disk

P388 IC₅₀ 28.5 ng/ml.

EXAMPLE 17 Mycalamide B 18-mono-trimethylsilylether

The compound of the formula: ##STR22##

Mycalamide B (0.7 mg) was dissolved in pyridine (0.1 mg) and cooled inice.BSA (N,O-bistrimethylsilyl acetamide) (0.1 ml) was added and thereaction was allowed to proceed for four minutes. Water (0.5 ml) wasthen added andthe mixture extracted with CH₂ Cl₂ (3×0.2 ml). The solventwas removed and the combined organic extracts were subjected to silicagelchromatography (200 mg Davisil, 150 A, 35-70 um), developed in stepsfrom hexane to ethyl acetate, A fraction (1 mg) eluted with 4:1hexane:ethyl acetate was pure mycalamide B 18-mono-trimethylsilyletherby PMR, being anoil having a molecular weight of 589 and a molecularformula C₂₈ H₅₁ NO₁₀ Si.

Spectral data:

HREIMS M+--CH₃ O 558.3048 (-9 ppm), M+--CH₃ OH 557.3022 (-0.4 ppm).

¹ H NMR (CDCl₃) 7.52(NH9,d,10.1), 5.79(H10,t,9.4), 5.13(10--OCH₂,d,6.9),4.86(4=CH₂,m), 4.85(10--OCH₂,d,6.9), 4.73(4=CH₂,t,1.8), 4.26(H7,broads), 4.19(H12,dd,6.2,10.0), 4.05(H2,dq,2.9,6.5), 3.98(7--OH,d,2.3),3.79(H11,dd,6.6,9.3), 3.64(H18,dd,2.6,11.4), 3.55(13--OCH₃,s),3.51(H18,dd,4.2,11.6), 3.41(H13,d,10.5), 3.33(H15,m), 3.31(6--OCH₃,s),3.23(17--OCH₃,s), 3.14(H17,m), 2.34(HS(eq),d,13.8), 2.26(H3,dq,2.6,7.1),2.18(H5(ax),td,2.4,13.9), 1.72(H16,ddd,1.8,9.8,13.8),1.5-1.6(H16,m)1.20(2--CH₃,d,6.6), 1.02(3--CH₃ d.7.2 ), 1.00(14--CH₃ (eq),s),0.87(14--CH₃ (ax),s), 0.13(18--OSi(CH₃)₃,s), ppm(couplings in Hz).

¹ H NMR (CD₂ Cl₂) 7.51(NH9,d,9.8), 5.77(H10,t,9.3),5.13(10--OCH₂,d,6.9), 4.86(4=CH₂,t,2.0), 4.84(10--OCH₂,d,7.0),4.70(4=CH₂,t,1.9), 4.24(H7,d,2.9), 4.16(H12,dd,6.3,9.8),4.02(H2,dq,2.8,6.6), 3.94(7--OH,d,2.8), 3.80(H11,dd,6.4,9.2),3.64(H18,dd,2.6,11.5), 3.54(13--OCH₃,s), 3.52(H18,dd,4.5,11.5),3.43(H13,d,10.3), 3.34(H15,dd,2.0,10.1), 3.28(6--OCH₃,s),3.20(16--OCH₃,s), 3.14(H17,m), 2.30(HS(eq),d,14.0), 2.27(H3,dq,2.4,6.6),2.17(H5(ax),td,2.2,14.0), 1.69(H16,ddd,2.0,9.7,14.2), 1.5(H16,m)1.21(2--CH₃,d,6.6), 1.02 (3--CH₃,d.7.2), 1.01(14--CH₃ (eq),s),0.87(14--CH₃ (ax),s),0.13(18--OSi(CH₃)₃,s), ppm(couplings in Hz).

¹³ C NMR (CD₂ Cl₂) 171.98(C8), 145.95(C4), 110.69(4=CH₂), 100.14(C6),86.59(10--O--CH₂), 79.82(C13), 78.92(C17), 76.25(C15), 74.36(C12),74.19(C10), 71.71(C7), 70.38(C11), 69.69(C2), 62.17(C18),61.60(13--O--CH₃), 56.69(C17--OCH₃), 48.48(6--O--CH₃), 41.57(C3),41.51(C14), 33.75(C5), 29.67(C16), 23.45(14--CH₃ (eq)), 17.89(2--CH₃),13.93(14 --CH₃ (ax),s),12.31(3--CH₃), -0.55(18--OSi(CH₃)₃).

AV assay: 4+, 4+, +*, @ 0.2 ng/disk; -, -, -@ 0.05 ng/disk

P388 IC₅₀ 0.08 ng/ml.

EXAMPLE 18 Mycalamide B bis-trimethylsilylether

The compound of the formula: ##STR23##

Mycalamide B (2.6 mg) was dissolved in pyridine (0.1 ml) and BSA(N,O-bistrimethylsilyl acetamide) (0.1 ml) and the reaction mixture wasstirred overnight at room temperature. Water (0.3 ml) was added and themixture extracted with CH₂ Cl₂ (3×0.2 ml). The solvent wasremoved andthe combined organic extracts were subject to silica gel chromatography(200 mg Davisil, 150 A, 35-70 um), developed in steps from hexane toethyl acetate. A fraction (3.1 mg) eluted with 4:1 hexane:ethyl acetatewas pure mycalamide B bistrimethylsilylether, being an oil having amolecular weight of 661 and a molecular formula C₃₁ H₅₉ NO₁₀ Si₂.

Spectral data:

HREIMS M+--CH₃ O 630.3318 (-28 ppm), M+--CH₃ OH 629.3418 (+0.5 ppm).

¹ H NMR (CDCl₃) 7.35(NH9,d,9.9), 5.78(H10,t,9.8), 5.13(10--OCH₂, d,7.0),4.82(10--OCH₂, D,6.9), 4.81(4=CH₂,t,1.9), 4.71(4=CH₂,t,2.0), 4.26(H7,s),4.20(12,dd,6.5,10.3), 3.90(H2,dq,2.7,6.6), 3.79(H11,dd,6.6,9.6),3.66(H18,dd,2.5,11.5), 3.55(13--OCH₃,s), 3.50(H18,dd,4.1,11.5),3.43(H13,d,10.3), 3.28(H15,m), 3.27(6--,&17--OCH₃,s), 3.18(H17,m),2.50(H5(eq),d,14.5), 2.34(H5(ax),td,2.0,14.5), 2.20(H3,dq,2.6,7.2),1.74(H16,ddd,1.8,9.9,14.0), 1.52(H16,m) 1.18(2--CH₃,d,6.6),1.00(3--CH₃,d.7.1), 0.98(14--CH₃ (eq),s), 0.87(14--CH₃ (ax),s),0.20(7--OSi(CH₃)₃,s), 0.12(18--OSi(CH₃)₃,s), ppm(couplings in Hz).

¹ H NMR (CD₂ Cl₂) 7.37(NH9,d,10.2), 5.75(H10,t,9.8), 5.15(10--OCH₂,d,7.0), 4.82(10--OCH₂,d,7.0), 4.82(4=CH₂,t,2.0), 4.68(4=CH₂,t,2.1),4.24(H7,s), 4.17(H12,dd,6.5,10.1), 3.87(H2,dq,2.7,6.6),3.80(H11,dd,6.5,9.5), 3.65(H18,dd,2.5,11.5), 3.54(13--OCH₃,s),3.49(H18,dd,4.3,11.5), 3.46(H13,d,10.1), 3.28(H15,m), 3.27 &3.25(6--&17--OCH₃,s), 3.18(H17,m), 2.48(H5(eq),d,14.3),2.34(H5(ax),td,2.1,14.5), 2.22(H3,dq,2.7,7.3),1.69(H16,ddd,2.1,9.7,14.0) 1.51(H16,m), 1.19(2--CH₃,d,6.6),1.01(3--CH₃,d.7.1), 1.00(14--CH₃ (eq),s), 0.87(14--CH₃ (ax),s),0.22(7--OSi(CH₃)₃,s), 0.13(18--OSi(CH₃)₃,s), ppm(couplings in Hz).

¹³ C NMR (CD₂ Cl₂) 170.90(C8), 147.32(C4), 109.60(4=CH₂), 99.60(C6),86.64(10--OCH₂), 79.72(C13), 78.94(C17), 77.38(C7), 76.02(C15),74.54(C12), 73.34(C10), 70.71(C11,broad), 69.69(C2), 62.20(C18),61.64(13--O--CH₃), 56.60(17--O--CH₃), 49.83(6--O--CH₃), 41.58(C3,C14),35.76(C5), 29.59(C16), 23.31(14--CH₃ (eq)), 17.85(2--CH₃), 13.70(14--CH₃(ax)), 11.90(3--CH₃), 0.28(7--OSi(CH₃)₃), -0.53(18--O(CH₃)₃).

AV assay: 4+, 4+, +*, @ 2 ng/disk; +, +, + @ 0.5 ng/disk

P388 IC₅₀ : 0.19 ng/ml.

EXAMPLE 19 4α-Dihydro mycalamide A

The compound of the formula: ##STR24##

Mycalamide A (5.5 mg), in methanol solution, was stirred with Adam'scatalyst (PtO₂,3mg) under H₂ for 1 hour, filtered and the solventremoved (5.4 mg). RPLC confirmed reduction and showed a 2:1 mix oftheepimers at C4. These were separated by prep RPLC giving two fractions(1.3 mg, 2.6 mg), the major one being found to be a complex mixture, butthe minor one being found to be a complex mixture, but the minor one tobethe pure 4α- epimer of dihydro mycalamide A by PMR, being an oilhaving a molecular weight of 505 and a molecular formula C₂₄ H₄₃NO₁₀.

Spectral data:

¹ H NMR (CDCl₃) 7.46(NH9,d,10.2), 5.85(H10,t,9.6), 5.13(10--OCH₂,d,6.9),4.87(10--OCH₂,d,6.9), 4.24(H7,s), 4.22(H12,dd,6.8,10.3),3.97(H2,dq,2.6,6.6), 3.83(H11,dd,6.4,9.7), 3.75(H17,m),3.6(H15,m,hidden), 3.56(13--OCH₃,s),3.55(H18,m,hidden),3.45(H13,d,10.2), 3.38(H18,dd,6.2,11.1),3.30(6--OCH₃,s), 2.15(H4,m), 1.65(H5(ax),m), 1.4(H3,m), 1.4(H3,m),1.18(2--CH₃,d,6.6), 0.98(14--CH₃ (eq),s), 0.88(4--CH₃,d,6.9),0.88(14--CH₃ (ax),s), 0.73(3--CH₃,d,7.0) ppm(couplings in Hz). NB.H5(eq), H3, H₂ 16 not observed.

AV assay: 4+, 4+, +*, @ 20 ng/disk; -, +, +* @ 5 ng/disk,

P388 IC₅₀ : 2.3 ng/ml.

EXAMPLE 20 Dihydro pseudomycalamide A

The compound of the formula: ##STR25##

4α-dihydromycalamide A in CDCl₃ solution was rapidly hydrolyxed on theaddition of D₂ O to give 4α-dihydro pseudomycalamide A cleanly, which isan oil having a molecular weight of 491 and a molecular formula C₂₃ H₄₁NO₁₀.

Spectral data:

DCIMS (NH₃) 509(9%,M+NH₄), 492(36%), 491(83%,M+NH₄ 30 --H₂ O), 474(16%,MH+--H₂ O), 292(17%), 262(23%).

¹ H NMR (CDCl₃) 7.49(NH9,d,9.9), 5.83(H10,t,9.9), 5.15(10--OCH₂,d,7.0),4.89(10--OCH₂,d,7.0), 4.24(H12,dd,6.8,10.5), 4.20(H2,dq,2.4,6.6),3.99(H7,s), 3.91(H11,dd,6.9,10.4), 3.74(H17,m), 3.59(H15,m,hidden),3.56(13--OCH₃,s), 3.55(H18,m,hidden), 3.48(H13,d,10.5),3.40(H18,dd,5.6,11.3), 2.2(H4,m), 1.67(H5(ax),t,13.2), 1.55(H₂16,m),1.42(H5(eq),dd,4.1,13.5), 1.4(H3,m), 1.06(2--CH₃,d,6.6),0.97(14--CH₃ (eq),s), 0.90(4--CH₃,d,6.9), 0.87(14 --CH₃ (ax),s),0.72(3--CH₃,d,7.0) ppm(couplings in Hz).

C NMR (CD₂ Cl₂) 97.09(C6), 87.04(10--OCH₂), 79.50(C13), 78.89(C15),74.51(C12), 73.20(C10), 73.11(C7), 72.06(C17), 71.24(C11), 69.83(C2),66.35(C18), 61.94(13--O--CH₃), 37.00(C3), 32.68(C5), 31.54(C16),29.00(C4), 22.94(14--CH₃ (eq)), 19.21(4--CH₃), 18.48(2--CH₃),13.22(14--CH₃ (ax)), 4.04(3--CH₃).

NB. C8, C14 not observed.

AV assay: 4+, 4+, +*, @ 200 ng/disk; 2+, +, +*, @ 50 ng/disk

P388 IC₅₀ : 10.5 ng/ml.

EXAMPLE 21 6-trideuteromethoxy mycalamide A

The compound of the formula: ##STR26##

Mycalamide A (1.1 mg) was dissolved in CD ₃ OD containing about 0.1%trifluoroacetic acid and allowed to react at room temperature overnight.PMR confirmed complete exchange to give pure C6--OCD₃ mycalamide A, anoil having a molecular weight of 506 and a molecular formula C₂₄ H₃₈ D₃NO₁₀.

Spectral data:

DCIMS (NH₃) 524(16%,M+NH₄), 491(14%), 490(22%), 489(81%,M+NH₄ +--CD₃OH), 472(16%,MH+--CD₃ OH), 278(16%), 245(19%), 244(96%).

EXAMPLE 22 4β-dihydro mycalamide A

The compound of the formula: ##STR27##

Mycalamide A (5.0 mg), in methanol solution, was stirred with Adam'scatalyst (PtO₂, 3 mg) under H₂ for 1 hour, filtered and the solventremoved (5.4 mg). RPLC confirmed reduction and showed a 2:1 mixofepimers at C4. These were separated by prep RPL giving two fractions(1.5 mg, 2.6 mg), the major one being found to be the 4β-epimer ofdihydromycalamide A by PMR, which is an oil with molecular weight 505and molecular formula C₂₄ H₄₃ NO₁₀.

AV assay: 3+, 3+, +*, @ 2 ng/disk, -, -, -, @ 0.5 ng/disk

P388 IC₅₀ : 0.8 ng.ml.

EXAMPLE 23 4β-dihydro pseudomycalamide A

The compound of the formula: ##STR28##

4β-dihydro mycalamide A dissolved in CD₂ Cl₂ immediately hydrolysedyielding 4β-dihydro pseudomycalamide cleanly, an oil with molecularweight 491 and molecular formula C₂₃ H₄₁ NO₁₀.

Spectral data:

¹ H NMR (CD₂ Cl₂) 7.25(NH9,d,10.0), 5.84(H10,t,10.0),5.17(10--OCH₂,d,7.0), 4.87(10--OCH₂,d,7.1), 4.44(H7,s),4.22(H12,dd,6.9,10.6), 4.08(H2,dq,3.0,6.5), 3.88(H11,dd,6.9,10.0),3.71(H17,m), 3.56(13--OCH₃,s), 3.55(H15,m,hidden), 3.53(H18,m),3.52(H13,d,10.4), 3.35(H18,dd,6.5,11.3), 1.9(H4,m), 1.55(H₂ 16,m),1.47(H3,m), 1.19(2--CH₃,d,6.6), 1.04(4--CH₃,d,7.0), 0.98(14--CH₃(eq),s), 0.88(4--CH₃,d,7.0), 0.87(14--CH₃ (ax),s), ppm(couplings in Hz).

NB: H₂ 5 not observed.

AV assay: 4+, 4+, +*, @ 20 ng/disk, 2+, 2+, +*, @ 5 ng/disk.

P388 IC₅₀ 4.2 ng/ml

EXAMPLE 24 4β-Dihydro mycalamide B

The compound of the formula: ##STR29##

Mycalamide B (5.5 mg ca), in methanol solution, was stirred with Adam'scatalyst (PtO₂,3 mg) under H₂ for 1 hour, filtered and the solventremoved (5.5 mg). RPLC confirmed reduction and showed a 3:2 mix oftheepimers at C4. These were separated by prep RPLC giving two fractions(1.7 mg, 2.7 mg), the major one being found to be the 4β-epimer ofdihydro mycalamide B and the minor one to be the 4α-epimer of dihydromycalamide B, both 90% pure by PMR. the 4β-epimer is an oil having amolecular weight of 519 and a molecular formula C₂₅ H₄₅NO₁₀.

Spectral data: ¹ H NMR (CD₂ Cl₂) 7.50(NH9,d,9.4), 5.76(H10,t,9.5),5.13(10--OCH₂,d,7.0), 4.84(10--OCH₂,d,7.0), 4.18(H7,s),4.18(H12,dd,6.5,10.2), 4.14(H2,dq,2.8,6.6), 3.83(H11,dd,6.7,9.5),3.60(H18,dd,3.4,11.8), 3.54(13--OCH₃,s), 3.45(H13,d,10.3),3.42(H18,dd,6.2,11.9), 3.41(H15,dd,2.4,9.5,partly hidden),3.28(17--OCH₃,s), 3.27(6--OCH₃, s), 3.24(H17,m), 1.69(H4,m),1.64(H5(ax),m), 1.5-1.6(H₂ 16,m), 1.53(H5(eq),m), 1.37(H3,m),1.19(2--CH₃,d,6.6), 1.18(4--CH₃,d,7.3), 1.00(14--CH₃ (eq),s),0.96(3--CH₃,d,7.1), 0.87(14--CH₃ (ax),s), ppm(couplings in Hz).

¹³ C NMR (CD₂ Cl₂) 171.97(C8), 100.30(C6), 86.72(10--OCH₂), 79.63(C13),79.23(C17), 75.60(C15), 74.58(C12), 74.15(C10), 73.04(C7), 70.92(C11),64.90(C2), 63.78(C18). 61.68(13--O--CH₃), 56.65(C17--OCH₃),47.90(6--OCH₃), 37.56(C3), 32.44(C5), 29.67(C16), 29.50(C4),23.20(14--CH₃ (eq)), 20.80(4--CH₃), 1828(2--CH₃). 13.03(3--CH₃). NB. C14, 14--CH₃ (ax) not observed.

AV assay: 4+, 4+, +*, @ 2 ng/disk; +, 2+, +*, @ 0.5 ng/disk

P388 IC₅₀ : 0.14 ng/ml.

EXAMPLE 25 4α-Dihydro mycalamide B

The compound of the formula: ##STR30##

The 4α-epimer of dihydro mycalamide B, prepared in the manner describedin Example 24, is an oil having a molecular weight of 519 and amolecular formula C₂₅ H₄₅ NO₁₀.

Spectral data:

¹ H NMR (CD₂ Cl₂) 7.53(NH9,d,9.3), 5.74(H10,t,9.4),5.12(10--OCH₂,d,7.0), 4.84(10--OCH₂,d,6.9), 4.21(H7,s),4.16(H12,dd,6.1,9.6), 3.99(H2,dq,2.4,6.6), 3.81(H11,dd,6.5,9.3),3.64(H18,m), 3.54(13--OCH₃,s), 3.45(H18,m), 3.44(H13,d,10.0),3.42(H15,m,hidden), 3.30(17--OCH₃,s), 3.29(6--OCH₃,s), 3.25(H17,m),2.18(H4,m), 1.5-1.6(H₂ 16,m), 1.20(2--CH₃,d,6.6), 1.01(14--CH₃ (eq),s),0.89(4--CH₃,d,6.3), 0.88(14--CH₃ (ax),s), 0.76(3--CH₃,d,7.1)ppm(couplings in Hz). NB: H₂ 5, H3 not observed.

EXAMPLE 26 4α Dihydro pseudomycalamide B

The compound of the formula: ##STR31##

4α-dihydro mycalamide B in CD₂ Cl₂ solution hydrolysed overnight to give4α-dihydro pseudo-mycalamide B which is an oil having a molecular weightof 505 and a molecular formula C₂₄ H₄₃NO₁₀.

Spectral data:

¹ H NMR (CD₂ Cl₂) 7.54(NH9,d,9.3), 5.67(H10,t,9.5),5.16(10--OCH₂,d,7.0), 4.99(6--OH,d,2.2), 4.85(10--OCH₂,d,7.0),4.21(H12,dd,7.1,10.0), 4.20(H2,dq,2.6,6.5), 3.95(H7,d,6.9),3.92(H11,dd,6.7,10.1), 3.82(7--OH,d,6.8), 3.55(13--OCH₃,s),3.54(H18,m,hidden), 3.47(H13,d,9.8), 3.44(H18,m,hidden), 3.37(H15,m),3.31(17--OCH₃,s), 3.3(H17,m), 2.23(H4,m), 1.69(H59(ax),dt,2.0,13.2),1.55(H₂ 16,m), 1.45(H3,m), 1.37(H59(eq),dd,3.9,13.2),1.07(2--CH₃,d,6.6), 0.98(14--CH₃ (eq),s), 0.92(4--CH₃,d,7.0),0.87(14--CH₃ (ax),s), 0.73(3--CH₃,d,7.1) ppm(couplings in Hz).

¹³ C NMR (CD₂ CL₂) 86.89(10--OCH₂), 79.30(C13), 79.04(C17), 75.28(C15),74.73(C12), 74.62(C10), 73.40(C7), 70.65(C11), 69.90(C2), 63.92(C18),61.84(13--O--CH₃), 56.79(C17--OCH₃), 41.84(C14), 32.27(C3), 32.84(C5),29.22(C16), 28.85(C4), 22.84(14--CH₃ (eq)), 19.20(4--CH₃),18.45(2--CH₃), 13.08(14--CH₃ (ax)), 3.95(3--CH₃). NB: C8, C6 notobserved.

AV assay: 4+, 4+, +*, @ 20 ng/disk; -, -, -, @ 5 ng/disk

P388 IC₅₀ : 3 ng/ml.

EXAMPLE 27 Pseudomycalamide B

The compound of the formula: ##STR32##

Mycalamide B (5.5 mg) was dissolved in CH₂ Cl₂ (0.5 ml) and stirred witha 1% aqueous solution of p-toluene sulfonic acid (0.2 ml) for48 hours atroom temperature. The organic extract was washed with water (3×0.3 ml),extracted in CH₂ Cl₂ and the solvent removed to give 5.1 mg of 85% purepseudomycalamide B, containing a small amount of mycalamide B by PMR.This compound is an oil having a molecular weight of 503 and a molecularformula C₂₄ H₄₁ NO₁₀.

Spectral data:

¹ H NMR (CD₂ Cl₂) 7.64(NH9,d,9.6), 5.68(H10,t,9.6),5.17(10--OCH₂,d,7.0), 4.88(4=CH₂,t,2.0), 4.86(10--OCH₂,d,7.1),4.74(4=CH₂,t,2.1), 4.22(H12,dd,6.7,10.5), 4.18(H2,dq,2.7,6.6),4.02(H7,s), 3.94(H11,dd,6.8,10.1), 3.56(13--OCH₃,s), 3.52(H13,d,10.5),3.47(H18,m), 3.4*(H18,m), 3.38*(H17,m), 3.33*(H15,m), 3.31(17--OCH₃,s),2.82(H5(ax),dt,2.2,13.8), 2.22(H3,dq,2.7,7.0), 2.12(H5(eq),d,13.7),1.5-1.65(H₂ 16,m), 1.09(2--CH₃,d,6.5), 1.01(3--CH₃,d,7.1), 0.98(14--CH₃(eq),s), 0.87(14--CH₃ (ax),s) ppm(couplings in Hz).NB: * exact positionsuncertain.

¹³ C NMR (CD₂ Cl₂) 174.44(C8), 147.33(C4), 109.79(4=CH₂), 97/76(C6),86.88(10--OCH₂), 79.26(C13), 78.95(C17), 75.04(C15), 74.71(C7*),74.54(C12*), 73.44(C10*), 70.61(C11), 68.95(C2), 63.97(C18),61.84(13--O--CH₃), 56.78(C17--OCH₃), 41.85(C14), 41.67(C3), 35.59(C5),28.82(C16), 22.84(14--CH₃ (eq)), 17.83(2--CH₃), 13.07(14--CH₃ (ax)),11.77(3--CH₃). NB: * assignments uncertain.

AV assay: 4+, 4+, +*, @ 5 ng/disk; -, -, -, @ 2 ng/disk

P388 IC₅₀ : 1.8 ng/ml.

EXAMPLE 28 Pseudomycalamide A

The compound of the formula: ##STR33##

Mycalamide A (2.5 mg) was dissolved in CH₂ Cl₂ (0.5 ml) and stirred witha 1% aqueous solution of p-toluene sulfonic acid (0.2 ml) for6 hours atroom temperature. The organic extract was washed with water (3×0.3 ml),extracted in CH₂ Cl₂ and the solvent removed to give 2.l mg of purepseudomycalamide A which is an oil having a molecular weight of 489 anda molecular formula C₂₃ H₃₉ NO₁₀.

Spectral data:

¹ H NMR (CD₂ Cl₂) 7.61(NH9,d,9.8), 5.83(H10,t,10.0),5.18(10--OCH₂,d,7.1), 4.88(10--OCH₂,d,7.0), 4.88(4=CH₂,t,2.0),4.75(4=CH₂,t,2.2), 4.24(H12,dd,6.8,10.5), 4.18(H2,dq,2.7,6.6),4.05(H7,broad d,6.1), 3.96(H11,dd,6.8,10.2), 3.73(H17,m),3.58(H18,m,hidden), 3.56(13--OCH₃,s), 3.53(H13,d,10.5),3.40(H18,dd,6.2,11.2), 2.82(H5(ax),td,2.0,13.8), 2.23(H3,dq,2.7,7.1),2.14(H5(eq),d,13.8), 1.5-1.6(H₂ 16,m), 1.09(2--CH₃,d,6.6), 1.01(3--CH₃,d,7.0), 0.99(14--CH₃ (eq),s), 0.88(14--CH₃ (ax),s) ppm(couplings in Hz).NB: H15 not observed.

¹³ C NMR (CD₂ Cl₂) 168.82(C8), 147.14(C4), 109.94(4=CH₂), 97.93(C6),87.21(10--OCH₂), 79.56(C13), 79.01(C15), 74.96(C12*), 74.74(C7*),73.42(C10*), 72.50(C11), 71.31(C7), 69.02(C2), 66.61(C18),61.88(13--O--CH₃), 42.00(C14), 41.65(C3), 35.80(C5), 31.72(C16),22.79(14--CH₃ (eq)), 17.84(2--CH₃), 13.19(14--CH₃ (ax)), 11.74(3--CH₃).NB: * assignments uncertain.

AV assay: 4+, 4+, +*, @ 200 ng/disk; 3+, 3+, +*, @ 50 ng/disk

P388 IC₅₀ : 23 ng/ml.

Biological Activity Assays Antitumor

Antitumor effectiveness of the compounds of the invention was evaluatedusing a standardized in vitro P388 mouse leukemia cell assay protocoldisclosed in U.S. Pat. No. 4,731,366 with the antitumor activity beingexpressed in IC₅₀ (concentration that results in 50% inhibition of cellreplication normalized to untreated cultures) determined with a log-logtransform of data and expressed as xx ng/ml. As an example, formycalamide A the P388 assay value was

    IC.sub.50 =1.6 ng/ml.

Antitumor effectiveness of mycalamide A was also evaluated using astandardized in vivo procedure by injecting mice with the test materialstarting 24 hours after P338 tumor implantation. A T/C value iscalculatedusing the formula % T/C=Median survival of test group/Mediansurvival of control group×100%, A T/C value above 130% is consideredsignificant. This resulted in the following test data:

    ______________________________________                                                     Median T/C %                                                     Dose mg/kg     1st series                                                                             2nd series                                            ______________________________________                                        0.02           Toxic    106                                                   0.01           116      156                                                   0.005          132      133                                                   0.0025         137      122                                                   0.0012                  117                                                   ______________________________________                                    

In vivo antitumor activity of mycalamide A was also tested with an B16murine melanoma assay resulting in the following data:

    ______________________________________                                        Dose mg/kg     Median T/C %                                                                              Cures                                              ______________________________________                                        0.5            Toxic       0                                                  0.25           2l4         l                                                  0.125          189         0                                                  0.0625         197         0                                                  ______________________________________                                    

It is apparent from the in vitro and in vivo data reported above, thatmycalamide A is effective for inhibiting or destroying tumors andtherefore in controlling diseases caused by or related to such tumors inhosts, including mammals, such as cancerous cachexia.

Antiviral

Anitviral effectiveness of mycalamide A against both Herpes simplex type1 virus (HVS--1) and Vesicular stomatitis virus (VSV) replicated in theCV--1 cell line. CV--1 is a fibroblast-like cell culture derived fromprimary African green monkey cells. A59 coronavirus tests were alsoconducted in vitro.

Antiviral activity is scored from 0 to +++, where +++=completeinhibition of plaque formation, ++=partial inhibition,+=less partialinhibition and and 0=no protection.

Cytotoxicity was determined in 24 well tissue culture plates of 16 mmdiameter. Discs are 6 mm in diameter. Zones of cytotoxicity greater than6mm are graded from 8 to 16 using only even numbers, in which, 0=nomacroscopic or microscope cytotoxicity, 16=100% or complete celldistruction, 8, 19, 12, 14=partial cytotoxicity, i.e., diameter of toxiczone including diameter of 6 mm. disc.

This antiviral assay work produced the following data for mycalamide A:

    ______________________________________                                                VSV      HSV-1      A-59                                              Dose (ug/well)                                                                          Cyt     AV     Cyt   AV   Cyt   AV                                  ______________________________________                                        40        16             16         50    3+                                  20        16             16         50    3+                                  2         16                        50    3+                                  0.2       12      2+     12    2+   0     2+/3+                               ______________________________________                                    

Mycalamide A was also tested in vivo with an A59 procedure in which fourweek old mice are inoculated intraperitoneally with 42 LD₅₀ doses ofvirus. Mycalamide A is administered on the same day and for 9consecutive days thereafter. Mice are held for observation for 14 daystotal. Mice inoculated with virus only are expected to die in 3 to 7days with a mortality rate of 80 to 100%. A reading of 0% mortality inmice receiving drug plus virus indicates protection from gross clinicaldisease and death. The results of this protocol are reported in thefollowing table:

    __________________________________________________________________________                    Cumu1ative Mortality Group                                              Drug conc.                                                                          Days after Infection                                          Mice      (mg/kg)                                                                             2 4  6   8   10  12  14                                       __________________________________________________________________________    Drug      4 1   0 0  100 100 100 100 100                                      Drug + virus                                                                            4 1   0 0  75  100 100 100 100                                      Drug      4 0.1 0 0  0   0   0   0   0                                        Drug + virus                                                                            4 0.1 0 0  0   0   0   0   0                                        Drug      4 0.01                                                                              0 0  0   0   0   0   0                                        Drug + virus                                                                            4 0.01                                                                              0 0  25  25  25  25  25                                       Virus only                                                                              8     0 63 88  100 100 100 100                                      Ribavirin (control)                                                           + virus   4 100 0 0  0   0   0   0   0                                        __________________________________________________________________________

The compounds of Examples 2-28 were subjected to antiviral assayevaluationsimilar to that reported above using polio vaccine virus TypeI (PVI) in place of VSV and using BSC cells rather than CV--1. In thisprotocol, bioassay discs (6.0 mm Schleicher and Schuell) are soaked withthe desiredamount of test solution, then air dried at ambienttemperature for 20 min. The discs are then pushed through the overlay tosit directly on the monolayer contained in a 16 mm diameter well andincubated in 5% CO₂ enriched atmosphere at 37 C for 24 hr. Wells areexamined, using an inverted microscope, for the size of antiviral and/orcytotoxic zones to evaluate the wells for the antiviral and cytotoxicityresults according tothe scale: -=no discernable antiviral or cytotoxiceffects, +=antiviral or cytotoxic zone 1-2 mm excess radius from disc,2+=zone of excess radius 2-4 mm, 3+=zone of excess radius above 4 mm,4+=effect over the whole well.

In reporting these values in Examples 2-28, the first value is for HSV,thesecond for PVI and the third for cytotoxicity (where +* impliesdiffuse cytotoxicity over the whole well). Thus, 4+, 4+, -, 5 ng/diskmeans 4+ forHSV, 4+ for PVI, negative for cytotoxicity with the drug at5 ng/disk.

DISCUSSION OF VARIABLES

Therapeutic and prophylactic application of the new compounds andcompositions containing them can be contemplated to be accomplished byanysuitable method and technique presently or prospectively known tothose skilled in the art. Further, the compounds of the invention haveuse as starting materials or intermediates for the preparation of otheruseful compounds and compositions.

The administration of the mycalamide compounds of the invention isuseful for treating antitumor and antiviral infections. Thus,pharmaceutical compositions containing compounds of the invention asactive ingredients are useful in prophylactic or therapeutic treatmentof humans or other mammals infected with or likely to be infected withtumors and virus.

The dosage administered will be dependent upon the identity of the tumororvirus infection, the type of host involved, its age, health, weight,kind of concurrent treatment, if any, frequency of treatment,therapeutic ratioand like considerations. Advantageously, dosage levelsof the administered active ingredients can be, for examples, dermal, 1to about 500 mg/kg; orally, 0.01 to 200 mg/kg; intranasal 0.01 to about100 mg/kg and aerosol 0.01 to about 50 mg/kg of animal body weight.

Expressed in terms of concentration, the active ingredient of theinventioncan be present in the new compositions for localized usedermally, intranasally, bronchially, intramuscularly, intravaginally,intravenously,or orally in a concentration of from about 0.01 to about50% w/w of the composition, and especially from about 0.1 to about 30%w/w of the composition.

The compositions of the invention are advantageously used in a varietyof forms, e.g., tablets, ointments, capsules, pills, powders, aerosols,granules and oral solutions or suspensions and the like containing theindicated suitable quantities of the active ingredient. Suchcompositions are referred to herein and in the accompanying claimsgenerically as "pharmaceutical compositions". Typically, they can be inunit dosage form,namely, in physically discrete units suitable asunitary dosages for human or animal subjects, each unit containing apredetermined quantity of active ingredient calculated to produce thedesired therapeutic or prophylactic effect in association with one ormore pharmaceutically acceptable other ingredients, e.g., diluent orcarrier.

Where the pharmaceutical compositions are aerosols, the activeingredients can be packaged in pressurized aerosol containers with apropellant, e.g.,carbon dioxide, nitrogen, propane, etc. with the usualadjuvants such as cosolvents, wetting agents, etc.

Where the pharmaceutical compositions are ointments, the activeingredient can be mixed with a diluent vehicle such as cocoa butter,viscous polyethylene glycols, hydrogenated oils and such mixtures can beemulsified if desired.

In accordance with the invention, pharmaceutical compositions comprise,as an active ingredient, an effective amount of one or more of the newcompounds and one or more non-toxic, pharmaceutically acceptableingredient. Examples of such ingredients for use in the compositionsinclude, ethanol, dimethyl sulfoxide, glycerol, silica, alumina, starch,calcium carbonate, talc, flour, and equivalent non-toxic carriers anddiluents.

In preferred embodiments for production of the new compounds byextraction from marine sponges, etc., suitable organic solvent systemsfor extractioncan be selected from methanol, ethyl acetate, acetone,diethyl ether, t-butyl methyl ether, ethanol, and isopropanol. Mixturesof two or more ofsuch solvents in various ratios and combinations areadvantageous.

Compounds of the invention are synthesized and/or isolated by variousfractionation and chromatographic techniques from the extracts obtainedasdisclosed. Preferred isolation procedures include variouschromatography techniques, e.g., countercurrent chromatography withsuitable columns, including multi-layer planetary coil columns. Avariety of solvents are available for use as single or mixed eluents,such as tetrahydrofuran, methanol, ethyl acetate, acetonitrile,n-propanol, n-butanol, water, and equivalent solvents. Furtherpurifications using such procedures may also be carried out on therecovered extractions. Preferred isolation techniques for furtherpurificaation include chromatographic operations such as high-pressure,liquid chromatography with suitable columns and suitable solvents.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A compound selected fromthe formula: ##STR34## wherein Z is ##STR35## R¹ and R² are methyl andR³⁻⁶ are the same or different and are hydrogen, lower alkyl, carboxylicacyl or lower tri-(lower alkyl) silyl.
 2. The compound of claim 1 of theformula: ##STR36##
 3. The compound of claim 1 of the formula: ##STR37##wherein Bz is bromobenzoyl.
 4. A pharmaceutical composition comprisingbetween about 0.01 to 50% by weight based on the total weight of saidcomposition as an active ingredient one or more compounds of claim 1 anda non-toxic carrier or diluent.
 5. A pharmaceutical compositioncomprising between about 0.1 to 25% by weight based on the total weightof said composition as an active ingredient the compound of claim 3 anda non-toxic carrier or diluent.
 6. A compound of the formula: ##STR38##wherein R³⁻⁶ are the same or different and are hydrogen, lower alkyl,carboxylic acyl or tri-(lower alkyl) silyl, andX is ═CH₂, ##STR39##
 7. Acompound of the formula: ##STR40## wherein R³⁻⁶ are the same ordifferent and are hydrogen, lower alkyl, carboxylic acyl or tri-(loweralkyl) silyl.
 8. A compound according to claim 6 of the formula:##STR41##
 9. A compound according to claim 6 of the formula: ##STR42##10. A compound according to claim 6 of the formula: ##STR43##
 11. Acompound according to claim 6 of the formula: ##STR44##
 12. A compoundaccording to claim 6 of the formula: ##STR45##
 13. A compound accordingto claim 6 of the formula: ##STR46##
 14. A pharmaceutical compositioncomprising between about 0.01 to 50% by weight based on the total weightof said composition as an active ingredient the compound of claim 5 anda non-toxic carrier or diluent.
 15. A pharmaceutical compositioncomprising between about 0.01 to 50% by weight based on the total weightof said composition as an active ingredient one or more of the compoundsof claim 11 and a non-toxic carrier or diluent.
 16. A pharmaceuticalcomposition comprising between about 0.01 to 50% by weight based on thetotal weight of said composition as an active ingredient one or more ofthe compounds of claim 7 and a non-toxic carrier or diluent.
 17. Apharmaceutical composition comprising between about 0.01 to 50% byweight based on the total weight of said composition as an activeingredient the compound of claim 8 and a non-toxic carrier or diluent.18. A pharmaceutical composition comprising between about 0.01 to 50% byweight based on the total weight of said composition as an activeingredient the compound of claim 9 and a non-toxic carrier or diluent.19. A pharmaceutical composition comprising between about 0.01 to 50% byweight based on the total weight of said composition as an activeingredient the compound of claim 10 and a non-toxic carrier or diluent.20. A pharmaceutical composition comprising between about 0.01 to 50% byweight based on the total weight of said composition as an activeingredient the compound of claim 11 and a non-toxic carrier or diluent.21. A pharmaceutical composition comprising between about 0.01 to 50% byweight based on the total weight of said composition as an activeingredient the compound of claim 12 and a non-toxic carrier or diluent.22. A pharmaceutical composition comprising between about 0.01 to 50% byweight based on the total weight of said composition as an activeingredient the compound of claim 13 and a non-toxic carrier or diluent.